The present invention relates to a process for the manufacture of polyguluronic acids having degrees of polymerization less than 20 and substantially free of mannuronic acid contamination.
Polyguluronic acids, because of their high affinity for calcium ions, are expected to have utility as scale inhibitors and scale deposit removers. The biodegradability of polyguluronic acids makes them particularly valuable with respect to environmental acceptability and waste disposal. Additionally, polyguluronic acid derivatives, in which a hydrophobic polymer is covalently attached to the reducing terminus of the polyguluronic acid, are useful as dispersants in pigment dispersed aqueous ink compositions for use in ink jet printing. Furthermore, polyguluronic acids, having low degrees of polymerization, have been shown to exhibit root-growth promoting activity in barley (M. Natsume et al., xe2x80x9cIsolation and Characterization of Alginate-derived Oligosaccharides with Root-Growth Promoting Activities,xe2x80x9d Carbohydrate Research, 258, 187-197 (1994)). They have also been shown to exhibit germination and shoot-elongation promoting activity in unhulled rice and tobacco callus (Y. Yonemoto et al., xe2x80x9cPromotion of Germination and Shoot Elongation of Some Plants by Alginate Oligomers Prepared with Bacterial Alginate Lyase,xe2x80x9d Journal of Fermentation and Bioengineering, 75, 68-70 (1993)). Based on studies of other polyuronic acids, low molecular weight polyguluronic acids might also be expected to exhibit antiviral, antitumoral, and plant-defense stimulating activities.
Alginic acids, from which polyguluronic acids can be obtained, are unbranched polymers of 1xe2x86x924 linked xcex1-L-guluronic acid (G) and xcex2-D-mannuronic acid (M) of varying proportions and sequence. A typical structure of an alginic acid molecule may be represented schematically as follows: 
As can be seen from the above structure, the distribution of monomers in alginic acid is not random and there is no regular repeat unit. Alginic acids are best described as block copolymers in which there are polyguluronic acid sequences (G-blocks), polymannuronic acid sequences (M-blocks), and sequences containing random arrangements of both guluronic acid and mannuronic acid (MG-blocks).
It is well known that alginates, which are salts of alginic acid, can be hydrolyzed and that the hydrolysis products can be separated to give two predominantly homopolymeric fractions, polyguluronic acid and polymannuronic acid. The most often cited procedure for the preparation of the sodium salt of polyguluronic acid is a heterogeneous acidic hydrolysis method disclosed in A. Haug et al., xe2x80x9cStudies on the Sequence of Uronic Acid Residues in Alginic Acid,xe2x80x9d Acta Chemica Scandinavica, 21, 691-704(1967). The acidic hydrolysis described therein requires that one part of sodium alginate be suspended in twenty parts of 0.3 M hydrochloric acid solution. Because alginic acid is insoluble in the strongly acidic solution, the hydrolysis is a heterogeneous reaction. The heterogeneous mixture is heated for 10+ hours at 100xc2x0 C. and then the solid is separated from the acidic solution by centrifugation or filtration. After the collected solid is dissolved in water by neutralizing with dilute sodium hydroxide solution, twenty parts of 0.3 M hydrochloric acid solution are added to the solution resulting in reprecipitation of the partially hydrolyzed alginic acid. The resulting heterogeneous mixture is heated for an additional 10+ hours at 100xc2x0 C. and the solid product is again separated from the acidic solution by centrifugation or filtration. The collected solid is dissolved in water by neutralizing with dilute sodium hydroxide solution and then sodium chloride and water are added to yield a solution which is 0.5 wt. % alginic acid and 0.1 M sodium chloride. An approximately equal volume of 0.025 M hydrochloric acid solution is added to the alginic acid salt solution until a pH value of 2.85 is obtained. The precipitated solid is separated from the acidic solution by centrifugation or filtration. The isolated solid is dissolved in water by neutralizing with dilute sodium hydroxide solution and then precipitated with excess ethanol. The precipitated solid is washed with ethanol, washed with ether, and dried. The sodium salt of the polyguluronic acid prepared by this heterogeneous acidic hydrolysis method has an average degree of polymerization between 15 and 20. The mannuronic acid content is between 5 and 15%, and the yield of product is between 15 and 20%.
During the course of the first step of the heterogeneous acidic hydrolysis, approximately 30% of the alginate goes into solution. An additional 15% of the original alginate goes into solution during the course of the second step. The insoluble fraction which is isolated after both steps contains both polyguluronic acid and polymannuronic acid. In the acidification of the dilute solution containing sodium salts of both polyguluronic acid and polymannuronic acid, polyguluronic acid is selectively precipitated.
Although the heterogeneous acidic hydrolysis method of A. Haug et al. is useful for laboratory scale preparations of polyguluronic acid, it would be difficult to implement on a larger scale, such as that which would be used in industrial production. This is because in the separation step of that method, the concentration of alginic acid is only a very dilute 0.25 wt. %. Additionally, the method has multiple steps and is complicated.
In the same reference as that describing the heterogeneous acidic hydrolysis method, a homogeneous acidic hydrolysis procedure is also reported. In that procedure, a 1 wt. % sodium alginate solution is mixed with an equal volume of a 0.025 M citrate buffer solution such that a combined solution having a pH value of 3.6 is obtained. The solution is boiled at reflux for 5+ hours. Although a method for isolating polyguluronic acids is not reported, presumably a method similar to that used in the heterogeneous acidic hydrolysis method can be used.
Although the homogeneous acidic hydrolysis method of A. Haug et al. may be useful for laboratory scale preparations of polyguluronic acid, it also would be difficult to implement on a larger scale. In that method, the concentration of sodium alginate in the hydrolysis step is only a very dilute 0.5 wt. %. Although one can easily conceive of increasing the concentration of sodium alginate, in practice this cannot be done. At concentrations slightly greater than 0.5 wt. %, sodium alginate does not remain soluble throughout the hydrolysis reaction in the range of pH values at which the acidic hydrolysis is effected. The species which precipitate in the course of the hydrolysis are incompletely hydrolyzed and, although rich in guluronic acid, they contain significant amounts of mannuronic acid. Furthermore, the incompletely hydrolyzed species have degrees of polymerization greater than 20. Because the homogeneous acidic hydrolysis conditions are milder than those described above in the heterogeneous acidic hydrolysis procedure, the incompletely hydrolyzed species are not further hydrolyzed after precipitating from solution.
Thus, there remains a need for a method of manufacturing polyguluronic acids which can be carried out on an industrial scale. Specifically, there remains a need for a hydrolysis process in which the concentration of alginic acid or alginate salt is greater than 5 wt. % throughout the process.
It is an object of the present invention to provide a practical process for the manufacture of polyguluronic acids having a degree of polymerization less than 20 and substantially free of mannuronic acid contamination.
The present inventor has found that alginate salts of organic bases are quite soluble in aqueous solution. Furthermore, the present inventor has found that these salts remain soluble in aqueous solution throughout a hydrolysis reaction in the range of pH values at which the hydrolysis is effected at a reasonably fast rate. The present invention has been made based on such findings.
According to one aspect of the present invention, there is provided a process for the manufacture of polyguluronic acids, having degrees of polymerization less than 20 and substantially free of mannuronic acid contamination, comprising the steps of:
(a) providing a solution containing 5 wt. % or more of alginic acid prepared by dissolving alginic acid by neutralization with an organic base;
(b) hydrolyzing the alginic acid to lower molecular weight components including polyguluronic acids while maintaining the pH of the solution on the acid side of neutrality;
(c) further acidifying the solution to selectively precipitate polyguluronic acids; and
(d) separating the polyguluronic acids from the acidified solution.